The invention generally relates to a confocal spectroscopic imaging system (CSIS) employing a confocal spectroscopic system (CSS) and/or a wavelength progammable light source (PLS). The components CSS and PLS can be used separately or together to create the CSIS. The invention particularly relates to a preferred implementation of the CSIS with a programmable array microscope (PAM) in which the patterns of illumination and detection are freely programmable.
Confocal microscopy based on point scanning systems with conjugate pairs of illumination and detection apertures is an effective tool for imaging a microscopic object to be investigated with direct optical sectioning. The discrete aperture spots are illuminated in the object plane of the microscope from which reflected or fluorescent light is observed through the conjugate detection apertures in an image plane. Commonly used confocal microscopes based on scanning systems with mechanically translated aperture disks (so-called Nipkow disks with a plurality of apertures) or with rotating mirrors being adapted to scan an object with a laser beam (Confocal Laser Scanning Microscopy, CSLM). Disk-based scanning systems have limitations as to a restriction of the illumination field, a degraded contrast and high intensity losses. Typically less than 3% of the aperture disk is transmissive since the spacing between the pinholes must be large to maintain the confocal effect. On the other hand, the CSLM suffers from a low duty cycle imposed by the sequential single point mode of data acquisition.
A modified spatially light modulated microscope has been described by M. Liang et al. in "Optics Letters" (vol. 22, 1997, p. 751-753) and in the corresponding U.S. Pat. No. 5,587,832. A two-dimensional spatial light modulator is formed by a digital micromirror device (in the following: "DMD") which reflects illumination light from a source (laser or white light source) to a probe and detection light from the probe to a two-dimensional detector. Each micromirror of the DMD is individually controlled so as to form an illumination and detection spot or not.
A general disadvantage of the prior art confocal microscopes concerns the detection of spectrally resolved images. The duty cycles and the illumination and detection intensities are limited so that a spectral imaging is not possible within practically interesting measuring times. Furthermore, excitation sources with rapid selection of large numbers (&gt;50) of multiplexed spectral elements at kHz-frequencies are not available.
Hadamard transform spectrometers (in the following: "HTS") are known which provide a positional and spectral selectivity on the basis of a detection through a so-called Hadamard encoding mask. Such spectrometers are described e. g. by R. M. Hammaker et al. in "Journal of Molecular Structure" (vol. 348, 1995, p. 135-138) or by P. J. Treado et al. in "Applied Spectroscopy" (vol. 44, 1990, p. 1-5, vol. 44, 1990, p. 1270-1275).
A Hadamard transform Raman microscope according to P. J. Treado et al. is shown in FIGS. 7A,B as HTS 70. FIG. 7A shows the arrangement of a Hadamard mask 72 in the detection path of a microscope 71. The Hadamard mask 72 is a linear encoding mask, and a spatial multiplexing of spectral measurments is obtained by driving the mask with a translation stage 73. The image obtained with the spectrograph 74 is decoded with a control computer 75. Details of the conventional microscope 71 used in the HTS 70 are shown in FIG. 7B.
The HTS according to P. J. Treado et al. has the following disadvantages. The illumination is restricted to a certain excitation wavelength without any controllability. The arrangement allows only Raman measurements. Because of the use of a conventional microscope, the generation of optical sections of an object under investigation without image processing is impossible. The mask is a non-programmable light modulator only.
Accordingly, the application of known HTS is generally restricted to optical detection paths or to acousto-optical measurements, Raman microscopy and conventional microscopy.
A confocal Raman microspectroscopy system is described by G. J. Puppels et al. in "Nature" (vol. 347, 1990, p. 301-303). Light scattered by an object under investigation is collected by an objective and coupled through a pinhole for confocal detection into a spectrometer. The detection through a pinhole represents an essential limitation of the measured light intensity so that the applicability of the known microspectroscopy system is restricted.
Confocal line scanning systems are commonly known. The work of P. A. Benedetti et al. in "Journal of Microscopy" (vol. 165, 1992, p. 119-129) discloses the use of a spectroscopic system with the confocal-line (CL) method. The CL technique has the following disadvantages. First, the formation of the line-shaped illumination needs extended optical components (slits, lenses, mirrors) with strict adjustment requirements. Second, the optical components are fixedly positioned, so that the scanning perpendicular to the line direction has to be performed by a mechanical object scanning table. Accordingly, the scanning speed is strongly restricted and particularly the spectral imaging is time consuming. Third, the illumination in this CL system is restricted to the use of one slit only. An operation with a multiplexing in a spatial domain is impossible.
Real-time confocal microscopy or imaging, in particular in the field of imaging biological objects like cells or parts thereof, calls for further improvements with regard to high contrast, sensitivity, detection speed and for an extended applicability of spectral imaging.